PROJECT SUMMARY/ABSTRACT There is an urgent need for an efficacious vaccine targeting HIV. Numerous potential HIV vaccines have been assessed, in particular those based on the Env viral coat proteins. However, results to date have been disappointing, with only one trial (RV144) showing significant efficacy. This is largely due to the extreme variability of the HIV Env protein. In addition, glycosylation of the Env protein is particularly complex, with up to 50% of the protein's weight due to covalently attached carbohydrates. This ?glycan shield? restricts accessibility of highly conserved epitopes and helps the virus evade the host immune system. Broadly neutralizing antibodies have been identified that recognize particular glycosylation patterns, and potential sites of vulnerability. An effective vaccine will likely use a combination of different Env proteins as a cocktail or in series for immunization rather than a single antigen, requiring production of large amounts of purified Env protein variants for testing. One challenge has been that the Env protein is unstable in its native trimer form. Also, yields of these proteins from cell culture are low, limiting research or potential future vaccine use. A system is therefore required that can produce larger amounts of the Env antigen variants in a stable form at a low cost. Plant-based systems can potentially supply large amounts of recombinant proteins compared to other hosts. They can produce kilogram quantities of antigens with low capital requirements and are capable of post- translational modifications such as glycosylation. With the appropriate choice of plant species and tissue for expression, processed material may be stable at ambient temperatures for years. The need to maintain a cold chain during storage is obviated and tissues such as maize grain are very low in interfering toxic compounds. These advantages may allow for inexpensive production of the large amounts of Env protein variants needed for development of an HIV vaccine. Vaccine candidates produced in plants have elicited an immune response and protection against pathogen challenge in animal trials and demonstrated safety and efficacy in human clinical trials. However, many practical barriers have prevented this approach from reaching widespread use. Encouragingly, a maize-based system has emerged as a commercially viable platform for recombinant protein production that can overcome these barriers. It has allowed some of the highest levels of expression reported in any plant system. In addition, targeting expression to the endoplasmic reticulum (ER) can result in a glycosylation pattern similar to that in mammalian cells. This may be particularly important for antigens such as the HIV Env proteins for which glycosylation is critical for development of neutralizing antibodies. The work described here proposes to develop a system for effective low-cost production of large amounts of HIV Env protein variants for near-term use as a research reagent and long-term eventual use in an HIV vaccine.